We have previously shown that prostacyclin activity is regulated b serum proteins. Serum protein stabilizes PGI2 probably through its binding to serum macromolecules. Defective PGI2 binding has been demonstrated in patients with thrombotic stroke and microvascular thrombosis. Physiological significance of abnormal binding activity remains unclear. Using a stable analog of PGI2, i.e. iloprost, we have shown that serum binding to PGI2 in a saturable manner with a KD of 7 x 10-5. Serum albumin binds to iloprost at a similar KD. Our previous studies have indicated that binding of PGI2 cannot be ascribed to albumin entirely. We plan to elucidate the mechanism by which binding occurs in serum. We plan to purify binding proteins from the serum, characterize the structural-functional relationship using novel probes. Once binding proteins are purified, we plan to produce monoclonal antibodies and develop sensitive assay for detection of the defect. The antibodies will be used for probing the structural abnormalities. Fluorescent probes such as 2, 6 dichloroaminophenol iloprost will be employed to determine binding abnormalities in thrombotic stoke. The purified protein or co-factor will be used to determine the interaction between binding proteins and the target cell, i.e. platelets. We plan to purify platelet PGI2, receptor and characterize the receptor protein and determine the active sites by novel probes. This will allow us to study the interaction between PGI2 binding proteins, PGI2 and receptors at the molecular level. PGI2 receptor defects in stroke patients will be studied. These studies will enhance our understanding of the molecular defects leading to stroke and provide a more direct means to develop new therapeutic strategies in controlling stroke and tissue typing.